Bacterial sepsis and related septic shock are frequently lethal conditions caused by infection which can result from surgery, trauma, and immune suppression related to cancer, transplantation therapy or other diseases. Gram-negative bacterial infections comprise the most serious infectious disease problem seen in hospitals today, now counting for thousands of infections yearly with a high overall mortality.
In prior decades, most infectious diseases contracted in hospitals were attributable to acute Gram-positive bacterial pathogens such as Staphylococcus and Streptococcus. However, in the last thirty years the incidence of nosocomial infections with Escherichia coli, Pseudomonas aeruginosa and other Gram-negative bacteria has risen steadily. The increase has been attributed to advances in medical treatment, resulting in prolonged survival of immunologically impaired hosts and the increased prevalence of wide spread antibiotic use in the hospital environment.
In addition to individuals receiving anti-cancer chemotherapy or immunosuppressive treatments following organ or tissue transplants, other individuals significantly at risk for Gram-negative bacterial infections include those with cystic fibrosis (CF) and burn victims. More than 90% of mortality in CF patients is the result of P. aeruginosa infections. Infections in immuno-compromised hosts typically exhibit resistance to many antibiotics, or develop resistance over the long course of the infection, making conventional treatment difficult. Natural selection for drug resistant bacteria by the extensive use of antibiotics has contributed to a Gram-negative bacteria involving into pathogens of major clinical significance.
A variety of factors contribute to the pathogenicity of Gram-negative bacteria. As described above, many Gram-negative bacteria are highly resistant to antibiotics. Additionally, many Gram-negative bacteria synthesize cellular and extra-cellular products to ensure infection of their hosts. Cellular products include lipopolysacciaride (LPS), pili, and alginate (in P. aeruginosa), a mucoid polysaccharide that is thought to protect the bacteria from phagocytosis. LPS is produced in a variety of forms, is highly immunogenic, mediates entry into eukaryotic cells and protects the bacteria from host defensives.
The LPS of P. aeruginosa and other Gram-negative bacteria is made up of a conserved structure that is widely shared among diverse Gram-negative bacterial genera. The core structure is comprised of three regions: (1) an O-antigen (O-polysaccharide comprised of repeating units of 3–5 sugars); (2) an inner and outer core; and (3) a lipid A region. The O-antigen is attached to the lipid A via the core region, and the lipid A portion is imbedded into the outer membrane of the organism where it serves as an anchor for the LPS. P. aeruginosa usually expresses two distinct types of LPS on its outer membrane, A-band and B-band. B-band LPS is the immunodominant antigen and is used to divide the organism into serogroups and subgroups. This is also seen in some other Gram-negative bacteria having LPS.
The lipid A portion of the LPS consists of short phosphorylated lipids acylated to a glucosamine disaccharide backbone and is thought to be the most toxic portion of the LPS. Also known as endotoxin A, it can overinduce the inflammatory response of the immune system, resulting in septic shock or even death. Septic shock causes a decrease in blood pressure, which can cause harm to the kidneys, lungs and gastrointestinal tract.
Pili are also an important virulence factor. Pili are hair-like fibers that extend outward from the bacterial cell and allow the bacteria to interact with surfaces, and are believed to be responsible for initial attachment of the bacteria to host cells. Without the ability to attach to host cells, bacteria are much less virulent. For example, Woods et al. (1980) demonstrated that the use of homologous anti-pilus antisera prevented attachment to buccal epithelial cells in challenge assays.
It has been shown that P. aeruginosa pilus is glycosylated (Castric, 1995, Microbiology, 141: 1247–1254); other type IV pilin bacteria are thought to be glycosylated as well. Structural analysis (Castric, et al., Journal of Biological Chemistry, 276:26479–26485) has shown that the pilin glycan is a trisaccharide that is identical to the O-antigen repeating unit of this microbe, and originates from the O-antigen biosynthetic pathway (unpublished observations). The pilin glycan is a major B-cell epitope of the pilus. Subcutaneous immunization of mice with pure glycosylated P. aeruginosa 1244 pili produced glycan (O-antigen)-specific antibodies. These antibodies recognized LPS from P. aeruginosa 1244 as determined by ELISA and Western blot (unpublished results). In addition, intranasal immunization of mice with glycosylated pili stimulated production of LPS-specific IgA in bronchial lavage and protected the animals against challenge with P. aeruginosa 1244. These results indicate that the P. aeruginosa pilin glycan produces an anti-LPS response.
Attachment of the O-antigen repeating unit to the pilus requires a functioning pilO gene (Castric, 1995, Microbiology, 141:1247–1254); the pilO protein in P. aeruginosa 1244 has been found to be extremely non-specific as to O-antigen repeating unit. For example (and as described more fully in the examples below), the gene cluster that codes for synthesis of the Escherichia coli O157 O-antigen was cloned in a broad-host-range cosinid and produced O157 LPS in P. aeruginosa 1244. The pilin sub-units produced by this strain were glycosylated with either O157 antigen or the host cell repeating unit (demonstrated in the examples presented below). This same nonspecificity was seen with a cloned heterologous P. aeruginosa O-antigen gene cluster.
As identified in numerous studies, the O-antigen repeating unit of LPS is the principal target of the immune response. However, developing a heterologous vaccine to O-antigens from multiple strains has proven difficult. For example, a study on the immune response in mice to high molecular weight O-antigens was performed with P. aeruginosa serogroup O2 strains (O2a–O2f). homologous O-antigens were more immunogenic in low doses, and elicited highly protective opsonic antibodies (Hatano and Pier, 1998). When vaccination was given as heterologous O-antigens, antibodies were generated to all strains, but cross-reactive opsonic antibodies were decreased, rather than enhanced among the strains tested (Hatano and Pier, 1998).
The use of an LPS-based vaccine has serious drawbacks, as this type of vaccine includes the toxic part (lipid A) of the LPS molecule, as well as the drawbacks discussed above regarding multivalent vaccines. There is a continued need for the development of compositions for eliciting an immune response in a vertebrate animal to Gram-negative bacterial infections, to assist the native immune response in overcoming such infections.